1. Field of the Invention
This invention relates to a novel gene sequence encoding barley endoxylanase. More specifically, the invention relates to a genomic nucleic acid sequence and the 62 kDa endoxylanase it encodes, which is useful to express enhanced amounts of endoxylanase in host cells, and particularly in plants transformed with the gene, permitting enhanced degradation of cell wall xylan.
2. Background of the Invention
Degradation of cell wall arabinoxylans in germinating cereal grains is mediated by the action of endoxylanase. Cell wall degradation is of particular importance in fermentation processes that rely on fermentable sugars and nutrients provided by degradation of cereal grains. For example, barley malt, wheat malt, cereal grain malt, and cereal adjuncts, such as grain or grits, commonly maize or rice, are primary sources of required nutrients in the brewing process. When brewing beer, the amount of starch and protein degradation during malting and mashing, as well as the subsequent separation of spent grain from wort extract, greatly impacts the quality of the final product.
Oligo and polysaccharides, derived from P-glucans and arabinoxylans, that are not well degraded and that remain in the wort extract cause significant difficulties during brewing. Solubilized non-starch polysaccharides (nsp) are the primary cause of undesirable wort viscosity. Insoluble arabinoxylans absorb significant amounts of water and form a thick layer on the filter during wort filtration. Thus, both insoluble arabinoxylan and soluble arabinoxylan and .beta.-glucan contribute to a reduced recovery of malt extract, impaired wort run-off during lautering (wort filtration), shortened half-life of wort filters, and haze formation in the beer product.
An analysis of the nsp content of fifteen commercial beers showed the arabinoxylan content ranged from 514 to 4211 .mu.g/ml. Beers with the highest xylan (arabinoxylan) content were premium beers to which cereal adjunct had been added. In particular, beers made from wheat malt, known to contain 12.6% w/w xylan, contained a high amount of xylan. In the study, the viscosity of beer significantly correlated with xylan content, as well as .beta.-glucan content, of the beer. (Schwarz and Han, 1995, "Arabinoxylan Content of Commercial Beers", Soc. Brewing Chemists, 53:157-159) It is therefore highly desirable to degrade arabinoxylan to short chain substituted arabinoxylo-oligomers early in the brewing process to avoid problems associated with non-degraded xylan.
Unlike .beta.-glucans, which are largely degraded during malting and to a lesser extent in mashing, xylan degradation is limited during the brewing process. In large part, the limited degradation of xylan is due to the unavailability of endoxylanase early in malting. As discussed above, high levels of residual xylan in malt or wort results in viscosity, haze and filtration problems. Therefore, it is highly desirable to enhance degradation of xylan during cereal grain processing, particularly during the early stages of brewing, to reduce residual xylan in the wort and final product. One way to enhance the degradation of xylan is to increase the availability of endoxylanase, for example, by enhancing expression of the endoxylanase gene in the grain during industrial malting or by adding endoxylanase enzyme to the wort.
Endo-.beta.-xylanase proteins previously purified from germinating barley include three 41 kDa isoforms of endoxylanase, XH1, XH2, and XH3, each with a pI of 5.2 (Slade, et al., 1989, Eur. J. Biochem. 185:533-539) and a 34 kDa endoxylanase with a pI of 4.6 (Benjavongkulchai, et al., 1986, Planta 169:415-419). A partial cDNA clone encoding the 41 kDa protein has been isolated (Banik, et al., 1996, Plant Molecular Biology, 31:1163-1172), however, the prior art does not indicate any protein has been expressed from this gene. As demonstrated in the Examples below, expression of this nucleic acid sequence resulted in little or no endoxylanase activity. (See Example 5 and FIGS. 18, 19 and 20.)
It would, therefore, be of great utility to isolate and characterize a gene encoding an active barley malt endoxylanase, and to express the endoxylanase gene in cereal grains during malting to bring about enhanced degradation of arabinoxylans in the malt and in the wort during mashing. Furthermore, expression of barley malt endoxylanase gene in an alternative host cell would provide quantities of purified enzyme for adding to the brewing process at the start of mashing to enhance degradation of arabinoxylan.